SCIENTIFIC OPINION. Abstract

Transcription

1 SCIENTIFIC OPINION ADOPTED: 26 October 2016 doi: /j.efsa Scientific Opinion on an application by DOW AgroSciences LLC (EFSA-GMO-NL ) for placing on the market the genetically modified herbicide-tolerant maize DAS for food and feed uses, import and processing under Regulation (EC) No 1829/2003 EFSA Panel on Genetically Modified Organisms (GMO), Hanspeter Naegeli, Andrew Nicholas Birch, Josep Casacuberta, Adinda De Schrijver, Mikołaj Antoni Gralak, Philippe Guerche, Huw Jones, Barbara Manachini, Antoine Messean, Elsa Ebbesen Nielsen, Fabien Nogue, Christophe Robaglia, Nils Rostoks, Jeremy Sweet, Christoph Tebbe, Francesco Visioli, Jean-Michel Wal, Fernando Alvarez, Michele Ardizzone, Antonio Fernandez Dumont, Yi Liu, Franco Maria Neri and Matthew Ramon Abstract Maize DAS was developed by direct Whiskers-mediated transformation to express the aryloxyalkanoate dioxygenase-1 (AAD-1) protein, conferring tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. The molecular characterisation of maize DAS did not raise safety issues. The agronomic, phenotypic and compositional characteristics of maize DAS tested under field conditions revealed no differences between maize DAS and its non-genetically modified (GM) comparator that would give rise to food and feed or environmental safety concerns. There were no concerns regarding the potential toxicity and allergenicity of the newly expressed protein AAD-1, and no evidence that the genetic modification might significantly change the overall allergenicity of maize DAS The nutritional characteristics of maize DAS are not expected to differ from those of non-gm maize varieties and no post-market monitoring of food/feed is considered necessary. Maize DAS is as nutritious as its non-gm comparator and other non-gm commercial varieties. There are no indications of an increased likelihood of establishment and spread of occasional feral maize DAS plants, unless these plants are exposed to the intended herbicides. However, this will not result in different environmental impacts compared to conventional maize. Considering the scope of the application, interactions with the biotic and abiotic environment were not considered an issue. Risks associated with the unlikely but theoretically possible horizontal gene transfer from maize DAS to bacteria were not identified. The post-market environmental monitoring plan and reporting intervals are in line with the scope of the application. In conclusion, the EFSA GMO Panel considers that the information available for maize DAS addresses the scientific comments raised by the Member States and that maize DAS , as described in this application, is as safe as the non-gm comparator and non-gm maize reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. Keywords: GMO, maize (Zea mays), DAS , herbicide tolerance, AAD-1, Regulation (EC) No 1829/2003 Requestor: Competent Authority of the Netherlands Question number: EFSA-Q Correspondence: EFSA Journal 2016;14(12):4633

2 Panel members: Andrew Nicholas Birch, Josep Casacuberta, Adinda De Schrijver, Mikołaj Antoni Gralak, Philippe Guerche, Huw Jones, Barbara Manachini, Antoine Messean, Hanspeter Naegeli, Elsa Ebbesen Nielsen, Fabien Nogue, Christophe Robaglia, Nils Rostoks, Jeremy Sweet, Christoph Tebbe, Francesco Visioli and Jean-Michel Wal. Acknowledgements: The Panel wishes to thank the following for the support provided: the members of the Working Group on Molecular Characterisation, Food and Feed Safety Assessment and Environmental Risk Assessment for the preparatory work on this scientific opinion. Suggested citation: EFSA GMO Panel (EFSA Panel on Genetically Modified Organisms), Naegeli H, Birch AN, Casacuberta J, De Schrijver A, Gralak MA, Guerche P, Jones H, Manachini B, Messean A, Nielsen EE, Nogue F, Robaglia C, Rostoks N, Sweet J, Tebbe C, Visioli F, Wal J-M, Alvarez F, Ardizzone M, Fernandez Dumont A, Liu Y, Neri FM and Ramon M, Scientific Opinion on an application by DOW AgroSciences LLC (EFSA-GMO-NL ) for placing on the market the genetically modified herbicide-tolerant maize DAS for food and feed uses, import and processing under Regulation (EC) No 1829/2003. EFSA Journal 2016;14(12):4633, 25 pp. doi: /j.efsa ISSN: European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority. This is an open access article under the terms of the Creative Commons Attribution-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited and no modifications or adaptations are made. The EFSA Journal is a publication of the European Food Safety Authority, an agency of the European Union. 2 EFSA Journal 2016;14(12):4633

3 Summary Following the submission of an application (EFSA-GMO-NL ) under Regulation (EC) No 1829/ by Dow AgroSciences LLC, the EFSA Panel on Genetically Modified Organisms (GMO Panel) was asked to deliver a scientific opinion on the safety of the herbicide-tolerant genetically modified (GM) maize (Zea mays L.) DAS (Unique Identifier DAS-4Ø278-9). The scope of application EFSA-GMO-NL is for import, processing, and food and feed uses of maize DAS within the European Union (EU), but excludes cultivation in the EU. The GMO Panel evaluated maize DAS with reference to the scope and appropriate principles described in its guidelines for the risk assessment of GM plants. The evaluation addressed the following components of the risk assessment: the molecular characterisation of the inserted DNA and analysis of the expression of the corresponding protein; the comparative analyses of compositional, agronomic and phenotypic characteristics; the safety of the newly expressed proteins and the whole food/feed with respect to potential toxicity, allergenicity and nutritional characteristics; the environmental risk assessment and the post-market environmental monitoring plan. Maize DAS was developed by direct Whiskers-mediated transformation of immature maize embryos. It expresses the aryloxyalkanoate dioxygenase-1 (AAD-1) protein, which confers tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. The molecular characterisation data established that maize DAS contains one functional insert consisting of an intact aad-1 expression cassette. No other parts of the plasmid used for transformation could be detected in maize DAS Bioinformatic analyses and genetic stability studies were performed and the results did not raise safety issues. The levels of the newly expressed protein present in maize DAS were obtained and reported adequately. Based on the agronomic and phenotypic characteristics of maize DAS tested under field conditions, no relevant differences were observed between maize DAS and its non-gm comparator, except for plant height and time to silking. None of the differences identified in forage and grain composition between maize DAS and the non-gm comparator required further assessment for food and feed safety. The safety assessment identified no concerns regarding the potential toxicity of the newly expressed AAD-1 protein in maize DAS , considering the results of a subacute 28-day toxicity study where no adverse effects were observed at the highest dose tested and considering the results on the structural and functional properties of the AAD-1 protein, including bioinformatic analyses. The GMO Panel did not identify indications of safety concerns regarding allergenicity or adjuvanticity with the AAD-1 protein or regarding the overall allergenicity of maize DAS Based on the comparative analysis, the nutritional characteristics of food and feed derived from maize DAS are not expected to differ from that of food and feed derived from non-gm maize varieties. The GMO Panel concludes that maize DAS is as safe and as nutritious as its non-gm comparator and the non-gm maize reference varieties. The GMO Panel considers that post-market monitoring of food/feed derived from maize DAS is not necessary, given the absence of safety concerns identified. Due to the low survival capacity of maize, the observed differences in plant height and time to silking are unlikely to change the fitness (e.g. survival, fecundity, competitiveness) or invasiveness characteristics of maize DAS plants. In the case of accidental release into the environment of viable seeds of maize DAS , there are no indications of an increased likelihood of establishment and spread of feral maize DAS plants, unless these plants are exposed to 2,4-D- or AOPP-containing herbicides. However, the GMO Panel is of the opinion that this will not result in different environmental impacts compared to conventional maize. Potential interactions with the biotic and abiotic environment were not considered to be an issue by the GMO Panel. Risks associated with an unlikely but theoretically possible horizontal gene transfer (HGT) from maize DAS to bacteria have not been identified. Therefore, considering the introduced traits, the outcome of the comparative analysis, the routes of exposure and the limited exposure levels, the GMO Panel concludes that maize DAS would not raise safety concerns in the event of accidental release of viable GM maize grains into the environment. The post-market environmental monitoring (PMEM) plan provided by the applicant is in line with the scope of the application and the requirements of the GMO Panel for PMEM of GM plants. The GMO Panel agrees with the reporting intervals proposed by the applicant in the monitoring plan. 1 Regulation (EC) No 1829/2003 of the European Parliament and of the Council of 22 September 2003 on genetically modified food and feed. OJ L 268, , p EFSA Journal 2016;14(12):4633

4 In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL , additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications. In conclusion, the GMO Panel considers that the information available for maize DAS addresses the scientific comments raised by the Member States and that maize DAS , as described in this application, is as safe as the non-gm comparator and non-gm maize reference varieties with respect to potential effects on human and animal health and the environment in the context of the scope of this application. 4 EFSA Journal 2016;14(12):4633

5 Table of contents Abstract... 1 Summary Introduction Background Terms of Reference as provided by the requestor Data and methodologies Data Methodologies Assessment Molecular characterisation Evaluation of relevant scientific data Transformation process and vector constructs Transgene constructs in the GM plant Information on the expression of the insert Inheritance and stability of inserted DNA Conclusion Comparative analysis Evaluation of relevant scientific data Choice of comparator and production of material for the comparative analysis Agronomic and phenotypic characteristics Compositional analysis Conclusion Food/feed safety assessment Evaluation of relevant scientific data Effects of processing Toxicology Animal studies with the food/feed derived from GM plants Allergenicity Nutritional assessment of GM food/feed Post-market monitoring of GM food/feed Conclusion Environmental risk assessment and monitoring plan Evaluation of relevant scientific data Environmental risk assessment Post-market environmental monitoring Conclusion Conclusions Documentation provided to EFSA References Abbreviations EFSA Journal 2016;14(12):4633

6 1. Introduction Maize DAS was developed to confer tolerance to 2,4-dichlorophenoxyacetic acid (2,4-D) and aryloxyphenoxypropionate (AOPP) herbicides. Tolerance to 2,4-D and AOPP herbicides is achieved by the expression of an aad-1 gene from Sphingobium herbicidovorans encoding the aryloxyalkanoate dioxygenase (AAD-1) enzyme Background On 11 November 2010, the European Food Safety Authority (EFSA) received from the Competent Authority of the Netherlands an application (Reference EFSA-GMO-NL ) for authorisation of genetically modified (GM) maize DAS (Unique Identifier DAS-4Ø278-9), submitted by Dow AgroSciences LLC within the framework of Regulation (EC) No 1829/2003 on GM food and feed. After receiving the application EFSA-GMO-NL , and in accordance with Articles 5(2)(b) and 17 (2)(b) of the Regulation (EC) No 1829/2003, EFSA informed the Member States and the European Commission, and made the summary of the application publicly available on the EFSA website. 3 EFSA initiated a formal review of the application to check compliance with the requirements laid down in Articles 5(3) and 17(3) of the Regulation (EC) No 1829/2003. On 20 January 2011 and 18 February 2011, EFSA received additional information requested under completeness check (on 20 December 2010 and 9 February 2011, respectively). On 11 March 2011, EFSA declared the application as valid in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003. EFSA made the valid application available to the Member States and the European Commission, and consulted nominated risk assessment bodies of the Member States, including national Competent Authorities within the meaning of Directive 2001/18/EC 4 following the requirements of Articles 6(4) and 18(4) of Regulation (EC) No 1829/2003, to request their scientific opinion. The Member States had 3 months after the date of receipt of the valid application (until 11 June 2011) to make their opinion known. On 20 May 2011, 7 September 2012, 21 May 2014, 19 June 2014, 18 November 2014, 19 February 2015, 27 February 2015, 27 March 2015, 14 April 2015, 24 April 2015, 17 July 2015 and 19 October 2015, the GMO Panel requested additional information from the applicant. The applicant provided the requested information on 01 February 2012, 29 January 2014, 23 July 2014, 29 July 2014, 16 December 2014, 12 March 2015, 7 April 2015, 27 April 2015, 1 June 2015, 24 August 2015, 31 March 2016 and 9 August The applicant also spontaneously provided additional information on 5 April 2011, 11 April 2012, 2 July 2012, 24 August 2012, 29 January 2014, 23 July 2014, 31 March 2016 and 9 August In giving its scientific opinion on maize DAS to the European Commission, the Member States and the applicant, and in accordance with Articles 6(1) and 18(1) of Regulation (EC) No 1829/2003, EFSA has endeavoured to respect a time limit of 6 months from the acknowledgement of the valid application. As additional information was requested by the EFSA Panel on Genetically Modified Organisms (GMO Panel), the time limit of 6 months was extended accordingly, in line with Articles 6(1), 6(2), 18(1) and 18(2) of Regulation (EC) No 1829/2003. According to Regulation (EC) No 1829/2003, this scientific opinion is to be seen as the report requested under Articles 6(6) and 18(6) of that Regulation and thus will be part of the EFSA overall opinion in accordance with Articles 6(5) and 18(5) Terms of Reference as provided by the requestor The EFSA GMO Panel was requested to carry out a scientific assessment of maize DAS for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003. Where applicable, any conditions or restrictions which should be imposed on the placing on the market and/or specific conditions or restrictions for use and handling, including post-market monitoring requirements based on the outcome of the risk assessment and, in the case of genetically modified organisms (GMOs) or food/feed containing or consisting of GMOs, conditions for the protection of particular ecosystems/environment and/or geographical areas should be indicated in accordance with Articles 6(5)(e) and 18(5)(e) of Regulation (EC) No 1829/ Dossier: Part I Section A5. 3 Available online: 4 Directive 2001/18/EC of the European Parliament and of the Council of 12 March 2001 on the deliberate release into the environment of genetically modified organisms and repealing Council Directive 90/220/EEC. OJ L 106, , p EFSA Journal 2016;14(12):4633

7 The GMO Panel was not requested to give an opinion on information required under Annex II to the Cartagena Protocol. Furthermore, the GMO Panel did not consider proposals for labelling and methods of detection (including sampling and the identification of the specific transformation event in the food/feed and/or food/feed produced from it), which are matters related to risk management. 2. Data and methodologies 2.1. Data In delivering its scientific opinion, the GMO Panel took into account application EFSA-GMO-NL , additional information provided by the applicant, scientific comments submitted by the Member States and relevant scientific publications Methodologies The GMO Panel carried out an evaluation of the scientific risk assessment of maize DAS for food and feed uses, import and processing in accordance with Articles 6(6) and 18(6) of Regulation (EC) No 1829/2003. The GMO Panel took into account the appropriate principles described in its guidelines for the risk assessment of GM plants and derived food and feed (EFSA, 2006a; EFSA GMO Panel, 2011a), the environmental risk assessment (ERA) of GM plants (EFSA GMO Panel, 2010c) and on the post-market environmental monitoring (PMEM) of GM plants (EFSA, 2006b; EFSA GMO Panel, 2011b). The comments raised by the Member States are addressed in Annex G of the EFSA overall opinion 3 and were taken into consideration during the evaluation of the risk assessment. 3. Assessment 3.1. Molecular characterisation Evaluation of relevant scientific data Transformation process and vector constructs Immature embryos of maize line Hi-II (a derivative of the A188 and B73 inbred maize lines) were transformed with an FspI fragment of plasmid pdas1740 by direct Whiskers-mediated transformation (Petolino et al., 2003; Petolino and Arnold, 2009). The DNA fragment to be transformed was obtained by a FspI digestion of plasmid pdas1740, which resulted in five fragments: a 6,236 bp fragment containing the aad-1 expression cassette, providing tolerance to 2,4-D and AOPP herbicides, two fragments containing a portion of the ampicillin resistance gene of the vector backbone (about 1 kb each), and two minor fragments of 9 bp. Fragments were separated by column chromatography and the 6,236 bp FspI fragment was isolated and used for transformation. 5 The 6,236 bp fragment consisted of the following genetic elements: the constitutive ZmUbi1 promoter from Zea mays; a synthetic, plant codon-optimised version of the aad-1 gene from soil bacterium S. herbicidovorans; the 3 0 untranslated region from the peroxidase gene from Z. mays (ZmPer 3 0 untranslated region (UTR)), used as a terminator. The expression cassette was flanked by identical matrix attachment regions (RB7 MAR v3 and RB7 MAR v4) from Nicotiana tabacum, in order to increase expression of the aad-1 gene. 6 Additional functional elements of the FspI digested plasmid pdas1740, not intended to be transferred into the maize genome, were plasmid backbone sequences of puc19, including an ampicillin resistance gene Transgene constructs in the GM plant Molecular characterisation of maize DAS was performed by Southern analysis, polymerase chain reaction (PCR) and DNA sequence analysis in order to determine insert copy number, size and organisation of the inserted sequences and to confirm the absence of plasmid backbone sequences. The approach used was acceptable both in terms of coverage and sensitivity. 7 5 Dossier: Part I Section C1. 6 Dossier: Part I Section C3. 7 Dossier: Part I Section D EFSA Journal 2016;14(12):4633

8 Southern analysis indicated that maize DAS contains a single insert, which consists of a single copy of the aad-1 expression cassette from pdas1740. The insert and copy number were confirmed by multiple restriction enzyme/probe combinations covering the 6,236 bp inserted fragment and flanking regions. PCR analyses confirmed the results obtained by Southern analyses. The absence of vector backbone sequences was demonstrated by Southern analysis using backbone-specific probes. The insert and 5 0 and 3 0 flanking regions (1,873 and 1,868 bp, respectively) of maize DAS were sequenced. The sequence of the insert confirmed the presence of a 4,816 bp fragment of pdas1740 which contains an intact aad-1 expression cassette, a 249 bp partial MAR v3 on the 5 0 terminus, and a 1,096 bp partial MAR v4 on the 3 0 terminus. Sequence analysis revealed that the aad-1 sequence in DAS maize is identical to the corresponding sequences in pdas1740, except for a single base pair change (from T to C) in the non-coding region of the 3 0 UTR of the aad-1 gene. 7 The possible interruption of known endogenous maize genes by the insertion in DAS maize was evaluated by bioinformatic analyses of the pre-insertion locus and of the genomic sequences flanking the insert. Comparison of the sequences of the flanking regions in DAS with that of the pre-insertion locus indicated a 21 bp insertion as well as a 2 bp deletion at the 5 0 junction in DAS A 1 bp insertion occurred at the 3 0 junction. 7 The bioinformatic analyses of the DAS flanking regions indicated that the insert in DAS most likely integrated into a region containing sequences showing similarity to a Grande retrotransposon. No evidence was found for the interruption of known endogenous gene in the maize genome. 8 The results of segregation (see Section ) and bioinformatic analyses established that the insert is located in the nuclear genome. 9 Updated bioinformatic analyses of the amino acid sequences of the newly expressed protein (AAD-1), according to EFSA guidance (EFSA GMO Panel, 2011a), did not indicate significant similarities to toxins and allergens. 10 In addition, updated bioinformatic analyses of the newly created open reading frames (ORFs) within the insert and at its junction sites did not indicate significant similarities to toxins and allergens Information on the expression of the insert AAD-1 protein levels were analysed by an enzyme-linked immunosorbent assay (ELISA) in material harvested from replicated field trials across eight locations in the USA during the 2009 growing season. 11 Samples analysed included leafs (V2 V4, V9 and R1), roots and pollen (R1), forage (R4), whole plants (R6), and grain at maturity both those treated and non-treated with quizalofop, 2,4-D or a combination of the two. The mean values and ranges of the AAD-1 protein levels in grains and forage are summarised in Table 1. Table 1: Protein expression data for AAD-1 in maize DAS (lg/g dry weight) grains and forage (number of grain and forage samples is 31 for unsprayed and 32 for herbicidetreated plants) Grains Untreated 2,4-D-treated Quizalofop-treated 2,4-D- and quizalofop-treated 3.95 (a) 1.03 (b) ( ) (c) ( ) Forage ( ) (ND 13.73) 2,4-D: 2,4-dichlorophenoxyacetic acid herbicide. (a): Mean. (b): Standard deviation. (c): Range ( ) ( ) ( ) ( ) Inheritance and stability of inserted DNA Genetic stability of the maize DAS insert was assessed by Southern analysis of genomic DNA from five different generations. 12 The restriction enzyme/probe combinations used were sufficient 8 Dossier: Part I Section D7; additional information: 9/8/ Dossier: Part I Section D2, D5; additional information: 9/8/ Dossier: Part I Section D2; additional information: 9/8/ Dossier: Part I Section D3. 12 Dossier: Part I Section D EFSA Journal 2016;14(12):4633

9 to conclude that all the plants tested retained the single copy of the insert and flanking regions, which were stably inherited in subsequent generations. Phenotypic stability was observed by segregation analysis of the quizalofop tolerance trait of maize DAS The results supported the presence of a single insertion, segregating in a Mendelian fashion Conclusion The molecular characterisation data establish that maize DAS contains a single insert consisting of one copy of the aad-1 expression cassette. Bioinformatic analyses of the sequences encoding the newly expressed proteins and other ORFs present within the insert or spanning the junctions between the insert and genomic DNA did not raise safety issues. The stability of the inserted DNA and of the introduced herbicide tolerance trait was confirmed over several generations. The levels of the AAD-1 protein were obtained and reported adequately Comparative analysis Evaluation of relevant scientific data Choice of comparator and production of material for the comparative analysis 13 Application EFSA-GMO-NL presents data on agronomic and phenotypic characteristics, as well as forage and grain composition, of maize DAS derived from field trials performed at eight sites in the USA in 2009 (Table 2). Table 2: Overview of comparative assessment studies with maize DAS provided in application EFSA-GMO- NL Study focus Study details Comparators Agronomic and phenotypic characteristics; composition Non-GM: non-genetically modified. Field trials, 2009, USA (eight locations) Non-GM comparator (XHH13 9 7SH382) Commercial reference varieties Six non-gm varieties Field trials for the agronomic, phenotypic and compositional assessment of maize DAS were conducted in major maize growing areas of the USA, 14 representing regions of diverse agronomic practices and environmental conditions. At each site, the following materials were grown in a randomised complete block design with four replicates: maize DAS not treated with the intended herbicides (DAS /untreated), DAS treated with 2,4-D (DAS /2,4-D), DAS treated with quizalofop (DAS /quizalofop), DAS treated sequentially with 2,4-D and quizalofop (DAS /2,4-D+quizalofop), the non-gm comparator (XHH13 9 7SH382), and three commercial non-gm maize reference varieties. All materials were treated (sprayed) with required maintenance pesticides (including conventional herbicides) according to local requirements. In total, six non-gm maize reference varieties were included across all the field trials sites. 15 The non-gm comparator XHH13 9 7SH382 had a genetic background similar to maize DAS as documented by the pedigree. 13 Statistical analysis of field trial data The statistical analysis of the agronomic, phenotypic and compositional data from the 2009 field trials followed the recommendations of the GMO Panel (EFSA GMO Panel, 2010a, 2011a). This includes, for each of the four treatments of maize DAS , the application of a difference test (between the GM maize and its non-gm comparator) and an equivalence test (between the GM maize and the set of non-gm commercial reference varieties). The results of the equivalence test are categorised into four possible outcomes (I IV, ranging from equivalence to non-equivalence) Dossier: Part I Section D7; additional information: 1/2/2012 and 23/7/ The sites were in Richland (IA), Carlyle (IL), Bradford (IL), Rockville (IN), La Plata (MO), Dudley (MO), York (NE) and Germansville (PA). 15 Dekalb 6343, Croplan 691, LG seeds 2597, Northup King 72-G8, Midland/Phillips 7B15 and Pioneer 32T In detail, the four outcomes are: category I (indicating full equivalence to the non-gm reference varieties); category II (equivalence is more likely than non-equivalence); category III (non-equivalence is more likely than equivalence); and category IV (indicating non-equivalence). 9 EFSA Journal 2016;14(12):4633

10 Agronomic and phenotypic characteristics 17 In total, 28 agronomic and phenotypic endpoints were evaluated. 18 For 16 agronomic/phenotypic endpoints 19 not fulfilling the requirements of the statistical tests described above (e.g. categorical endpoints), a non-parametric test of difference (Wilcoxon signed-rank test) was performed. No statistically significant differences were identified for any of the endpoints. Of the 12 endpoints analysed with parametric statistics, the test of equivalence could not be applied to plant height because of the very small variation among the non-gm reference varieties. Plant height was found significantly different between maize DAS and the non-gm comparator for one of the four treatments (2,4-D+quizalofop). 20 The combination of the test of difference and the test of equivalence could be applied to the remaining 11 endpoints, with the following results: The test of difference between phenotypic and agronomic characteristics of maize DAS / untreated and the non-gm comparator identified statistically significant differences for two endpoints (early stand count at V1 and time to silking). The test of equivalence between maize DAS /untreated and the non-gm maize reference varieties indicated that early stand count at V1 fell under equivalence category I and time to silking fell under equivalence category II. For DAS /2,4-D, statistically significant differences were identified for seven endpoints (early stand count at V1 and V4, final stand count, yield, pollen colour at 30 min, pollen shape at 30 and 60 min). The test of equivalence showed that all these endpoints fell under equivalence category I. For DAS /quizalofop, statistically significant differences were identified for three endpoints (yield, pollen colour at 30 min, and pollen shape at 60 min). The test of equivalence showed that all these endpoints fell under equivalence category I. For DAS /2,4-D+quizalofop, statistically significant differences were identified for five endpoints (yield, pollen colour at 30 and 60 min, pollen shape at 30 and 60 min). The test of equivalence showed that all these endpoints fell under equivalence category I. Time to silking for DAS /untreated was found significantly different than the non-gm comparator and fell under equivalence category II. 21 Plant height for DAS /2,4-D+quizalofop was significantly different than the non-gm comparator and could not be categorised for equivalence. The results for time to silking and plant height are further assessed for their potential environmental impact in Section Compositional analysis 22 Maize forage and grain harvested from the field trials in the USA in 2009 were analysed for 82 compounds (nine compounds for forage 23 and 73 compounds for grain 24 ). The compounds included the key constituents recommended by OECD (2002). 17 Dossier: Part I Sections D4, D7; additional information: 2/7/2012, 24/8/2012, 29/1/2014, 23/7/2014 and 7/4/ Early stand count (V1 and V4), seedling vigour, plant vigour (injury from each of five herbicide applications), time to silking, time to pollen shed, pollen viability (pollen shape and colour, both measured at 0, 30, 60 and 120 min), plant height, ear height, stalk lodging, root lodging, final stand count, days to maturity, stay green, disease incidence, insect damage, and yield. 19 Seedling vigour, plant vigour (five endpoints), pollen shape (0 and 120 min), pollen colour (0 and 120 min), stalk lodging, root lodging, days to maturity, stay green, disease incidence, and insect damage. 20 Mean values (cm): the non-gm comparator: 280.4; DAS /2,4-D+quizalofop: Mean values (heat units): non-gm comparator: 1262; DAS /untreated: Dossier: Part I Section D7; additional information: 1/2/2012, 11/4/2012, 2/7/2012, 24/8/2012, 29/1/2014, 23/7/2014, 12/3/2015 and 7/4/ Proximates (crude protein, total fat, ash and moisture), carbohydrates by calculation, fibre fractions (acid detergent fibre (ADF) and neutral detergent fibre (NDF)), calcium and phosphorus. 24 Proximates (crude protein, total fat, ash, and moisture), fibre fractions (ADF, NDF and total dietary fibre), amino acids (alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), fatty acids (caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), myristoleic acid (C14:1), pentadecanoic acid (C15:0), pentadecenoic acid (C15:1), palmitic acid (C16:0), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), c-linolenic acid (C18:3), arachidic acid (C20:0), eicosenoic acid (C20:1), eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3), arachidonic acid (C20:4) and behenic acid (C22:0).), 10 minerals (calcium, phosphorous, potassium, sodium, iron, copper, magnesium, manganese, selenium and zinc), eight vitamins (vitamin B1 (thiamine), vitamin B2 (riboflavin) vitamin B3 (niacine), vitamin B6 (pyridoxine), vitamin B9 (folic acid), b-carotene, vitamin C and vitamin E), and other compounds (inositol, furfural, p-coumaric acid, ferulic acid, phytic acid, trypsin inhibitor and raffinose) EFSA Journal 2016;14(12):4633

11 The GMO Panel considered the studies performed by the applicant concerning the substrate specificity of the newly expressed protein AAD-1, which showed that AAD-1 appears to be capable of oxidising the maize endogenous compounds trans-cinnamic acid and indole-3-acetic acid. 25 The GMO Panel noted that phenylalanine and coumaric acid, involved in the phenylpropanoid biosynthesis and metabolically related to trans-cinnamic acid, were included in the spectrum of compositional parameters. Seventeen grain constituents 26 with more than 50% of the observations below the limit of quantification were excluded from the statistical analysis. Of the remaining 65 compounds, the test of equivalence could not be applied to one forage constituent (total fat) and to five grain constituents 27 because of the very small variation among the non-gm reference varieties. Among those six constituents, arginine content was found significantly different than the non-gm comparator (Table 3). Table 3: Endpoint Glycine (% AA) Leucine (% AA) Methionine (% AA) Phenylalanine (% AA) Carbohydrates (% DM) Crude protein (% DM) b-carotene (mg/kg DM) Arginine (% AA) Compositional endpoints that are further considered based on the results of the statistical analysis: means (for the GM maize and the non-gm comparator) and equivalence limits (from the non-gm reference varieties) estimated from field trials data (USA 2009, Table 2) Non-GM comparator XHH13 3 7SH382 Maize DAS Untreated (a) 2,4-D (b) Quizalofop (c) 2,4-D+ quizalofop (d) Equivalence limits from non-gm reference varieties * 3.55* 3.48* 3.51* (3.54, 4.06) * 13.26* 13.40* 13.32* (12.18, 13.33) * 1.75 (1.77, 2.2) * 5.44* 5.41 (4.98, 5.32) * 82.8* 82.74* 82.91* (83, 86.75) * 11.67* 11.69* 11.51* (8.99, 11.23) * 2.88* 2.95* 2.80* (0.61, 1.48) * 4.51* 4.4* 4.48* For the GM maize, significantly different entries are marked with an asterisk, while the outcomes of the test of equivalence are differentiated by greyscale backgrounds: white (for equivalence categories I II and for arginine, for which the test was not performed), light grey (equivalence category III) and dark grey (equivalence category IV). % AA: percentage total amino acids; DM: dry matter; : the test of equivalence was not applied because of the small variation among the non-gm reference varieties. (a): maize DAS given no target herbicide treatment. (b): maize DAS treated with 2,4-D. (c): maize DAS treated with quizalofop. (d): maize DAS treated sequentially with 2,4-D and quizalofop. For the remaining 59 endpoints, the results of the difference and equivalence tests were as follows: The test of difference between maize DAS /untreated and the non-gm comparator identified statistically significant differences for 20 constituents 28 (16 in grain and four in 25 Dossier: Part I Section D7.8.1; additional information: 1/2/2012 and 1/6/ Caprylic acid (C8:0), capric acid (C10:0), lauric acid (C12:0), myristic acid (C14:0), myristoleic acid (C14:1), pentadecanoic acid (C15:0), pentadecenoic acid (C15:1), palmitoleic acid (C16:1), heptadecanoic acid (C17:0), heptadecenoic acid (C17:1), gamma-linolenic acid (C18:3), eicosadienoic acid (C20:2), eicosatrienoic acid (C20:3), arachidonic acid (C20:4) and behenic acid (C22:0), sodium and furfural. 27 Inositol, phytic acid, ash, selenium and arginine. 28 Forage: carbohydrates, crude protein, calcium and phosphorus; grain: carbohydrates, crude protein, aspartic acid, glutamic acid, glycine, leucine, proline, threonine, valine, trypsin inhibitor, stearic acid (C18:0), moisture, calcium, niacin, b-carotene and ascorbic acid EFSA Journal 2016;14(12):4633

12 forage). The test of equivalence between maize DAS /untreated and the set of non-gm maize reference varieties indicated that the level of 15 of the 20 constituents fell under equivalence category I or II, while the level of five grain constituents fell under equivalence category III or IV (Table 3). For DAS /2,4-D, statistically significant differences were identified for 20 constituents 29 (16 in grain and four in forage). The test of equivalence showed that the level of 16 of the 20 constituents fell under equivalence category I or II, while the level of four grain constituents fell under equivalence category III or IV (Table 3). For DAS /quizalofop, statistically significant differences were identified for 26 constituents 30 (21 in grain and five in forage). The test of equivalence showed that the level of 19 of the 26 constituents fell under equivalence category I or II, while the level of seven grain constituents fell under equivalence category III or IV (Table 3). For DAS /2,4-D+quizalofop, statistically significant differences were identified for 14 constituents 31 (11 in grain and three in forage). The test of equivalence showed that the level of 10 of the 14 constituents fell under equivalence category I or II, while the level of four grain constituents fell under equivalence category III or IV (Table 3). The GMO Panel considered the results for the two compounds metabolically related to transcinnamic acid; no differences were identified for coumaric acid, and the difference identified for phenylalanine (Table 3) does not pose a safety concern. Therefore, it is unlikely that the AAD-1 enzyme interacts with endogenous plant metabolism in a way which may pose a safety concern. The GMO Panel assessed all the compositional differences between maize DAS and the non-gm comparator. After considering the biological role of the compounds and the magnitude and direction of the changes observed, the GMO Panel did not identify any need for further assessment with regard to food and feed safety Conclusion The GMO Panel concludes that none of the differences identified in grain and forage composition between maize DAS and the non-gm comparator, and none of those identified in the agronomic and phenotypic characteristics, needs further assessment regarding food and feed safety. Based on the agronomic and phenotypic characteristics of maize DAS tested under field conditions, none of the differences identified between maize DAS and the non-gm comparator are considered relevant except for plant height and time to silking, which are further assessed for their potential environmental impact in Section Food/feed safety assessment Evaluation of relevant scientific data Effects of processing 32 Processed products Based on the outcome of the comparative assessment (Section 3.2), processing of maize DAS into food and feed products is not expected to result in products different from those of commercial non-gm maize varieties. Newly expressed proteins 33 a) Effect of temperature on AAD-1 protein. The thermal stability of the bacterial AAD-1 protein was evaluated by heating protein solutions for 30 min at 50, 70 and 95 C in a buffer solution. At all heating 29 Forage: carbohydrates, crude protein, calcium and phosphorus; grain: carbohydrates, crude protein, glycine, leucine, phenylalanine, threonine, raffinose, stearic acid (C18:0), arachidic acid (C20:0), moisture, calcium, iron, phosphorus, niacin, b-carotene and ascorbic acid. 30 Forage: ash, carbohydrates, crude protein, calcium and phosphorus; grain: carbohydrates, crude protein, alanine, cystine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, threonine, tryptophan, valine, trypsin inhibitor, stearic acid (C18:0), calcium, phosphorus, zinc, niacin, b-carotene and ascorbic acid. 31 Forage: carbohydrates, crude protein and phosphorus; grain: carbohydrates, crude protein, aspartic acid, glycine, leucine, threonine, stearic acid (18:0), moisture, calcium, b-carotene and niacin. 32 Dossier: Part I Section D Dossier: Part I Section D7.8.1; additional Information: 23/7/2014 and 27/4/ EFSA Journal 2016;14(12):4633

13 conditions, the enzymatic activity of the protein was reduced to less than 3%. Only 1% of its immunoreactivity, as measured by a polyclonal antibody sandwich ELISA, was still observed at the temperatures tested. The molecular mass of the AAD-1 protein was unchanged, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). b) Effect of ph on the AAD-1 protein. The effect of ph on the bacterial AAD-1 protein in vitro activity was determined using 2,4-D as a substrate, revealing a ph optimum of 7.9 with progressive loss of activity at decreasing and increasing ph values. SDS-PAGE indicated that the AAD-1 protein concentration remained consistent over this ph range, suggesting that loss of enzymatic activity resulted from disruption of enzyme structure and not degradation of the AAD-1 protein Toxicology Maize DAS expresses the new protein AAD-1 (Section 3.1.1). Proteins used for safety assessment Given the technical restraints in producing large enough protein quantities for safety testing from plants, AAD-1 protein was recombinantly produced in Pseudomonas fluorescens. The purified protein from the bacterial source and from maize DAS were characterised and compared in terms of their physicochemical, structural and functional properties. AAD-1 characterisation and equivalence 34 SDS-PAGE showed that both plant- and microbe-derived AAD-1 proteins migrated close to the expected molecular weight of ~ 33 kda and were comparably immunoreactive to AAD-1 protein specific antibodies as shown by western blot analysis. In addition, glycosylation detection analysis demonstrated that the plant- and microbe-derived AAD-1 proteins were not glycosylated. Amino acid sequence analysis by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and electrospray ionisation liquid chromatography/mass spectrometry (ESI-LC/MS) showed that both proteins matched the expected AAD-1 sequence. These data also revealed that the N-terminal methionine of both proteins was truncated. Additional variants were observed for the plant protein with another three short truncations up to alanine 4. A small portion of the plant protein (3%) was also N-acetylated. The C-termini were identical for both proteins and fully matched the theoretical AAD-1 sequence. Functional equivalence was demonstrated by a biochemical in vitro activity assay which showed that both proteins had comparable activity and specificity for the intended herbicides. Furthermore, the microbial AAD-1 protein was screened for its ability to utilise certain endogenous plant substrates. The data demonstrated that AAD-1 activity is unlikely to have a metabolic impact within transgenic plants. In addition, to justify the appropriateness of the microbial recombinant AAD-1 protein used in a 28-day oral toxicity study in mice, 35 equivalence with the DAS expressed AAD-1 protein was demonstrated by MS analysis and enzymatic activity assay. Based on these data, the GMO Panel accepts the use of the microbial recombinant AAD-1 proteins for the safety studies. Toxicological assessment of newly expressed protein The AAD-1 protein has never been assessed by the GMO Panel. a) Bioinfomatic studies 36 Bioinformatic analyses of the amino acid sequence of the AAD-1 protein expressed in maize DAS revealed no relevant similarities to proteins known to be toxic to humans and animals (Section ). b) In vitro degradation studies 37 The resistance to degradation by pepsin of the bacterial AAD-1 protein was investigated in solutions at ph ~ 1.2. The integrity of the test protein in probes taken at various time points was analysed by SDS-PAGE followed by protein staining or western blot. The AAD-1 protein was degraded by pepsin within 30 sec. 34 Dossier: Part I Section D7.8.1; additional information: 1/2/2012, 1/6/2015 and 9/8/ Additional information: 9/8/ Dossier: Part I Section D7.8.1; additional information: 29/1/ Dossier: Part I Section D7.9.1; additional information: 29/7/ EFSA Journal 2016;14(12):4633

14 c) Acute oral toxicity testing 38 The bacterial-derived AAD-1 protein was administrated by oral gavage at a dose of 2,000 mg/kg body weight (bw) to male and female Crl:CD1(ICR) mice. No effects related to the AAD-1 protein were observed. The GMO Panel is of the opinion that acute toxicity testing of the newly expressed proteins is of little additional value for the risk assessment of the repeated consumption of food and feed from GM plants by humans and animals. d) 28-day repeated dose toxicity studies 39 The applicant provided a 28-day oral repeated dose toxicity study in mice to investigate the potential toxicity of the AAD-1 protein. However, the GMO Panel did not consider the overall study design adequate to identify the potential hazard of the AAD-1 protein, because of the low doses of AAD-1 protein tested (highest dose level approximately 45 mg/kg bw per day) and the limited number of animals used in treatment groups (5 per sex per group) to ensure an adequate statistical power (EFSA GMO Panel, 2011a). Moreover, the GMO Panel noted that analyses of coagulation were not performed. The GMO panel requested a 28-day toxicity study in rodents, to support the safety assessment of the AAD-1 protein, with a sufficient number of animals, selecting the doses according to the OECD TG 407 in order to induce adverse effects at the highest dose, or following a limit test approach, if toxicity is not expected. Following the EFSA request, the applicant provided a new 28-day oral repeated dose toxicity study in mice, conducted in accordance with the OECD TG 407 and in compliance with the principles of Good Laboratory Practice (GLP). Groups of singly caged Crl:CD1(ICR) mice (12 per sex per group) were administered by gavage the AAD-1 protein, at a targeted dose of 1,000 mg/kg bw per day (AAD-1 protein group); the vehicle alone (vehicle control group) and bovine serum albumin (BSA) at a targeted nominal dose of 1,000 mg/kg bw per day (BSA control group). The ADD-1 and BSA protein formulations were prepared daily; for the AAD-1 protein group, the concentration of the AAD-1 protein in the test material (63.5%) was taken into account to meet the targeted dose. Feed and water were provided ad libitum. Animals were checked daily for mortality and general clinical signs; detailed clinical observations were conducted on all animals pretreatment and then weekly. Ophthalmoscopy was carried out before the start and at the end of the treatments. Body weights were recorded on test day 1, 2, 3, 4, 8, 15, 22 and 29 (terminal body weight) and body weight gains were calculated relative to test day 1. Feed consumption was determined on test days 1 2, 2 3, 3 4, 4 8, 8 15 and At the end of the treatment period, haematological and clinical chemistry analyses were performed. All animals were sacrificed and underwent a complete necropsy examination with selected organs weighed. Organs and tissues from all animals were subjected to a comprehensive histological examination. The GMO Panel noted that the AAD-1 protein formulations were prepared from the powdered test material stored at approximately 4 C until use. Stability tests on the powdered test material (i.e. the lyophilized AAD-1 protein) were not performed as part of this study and, according to the study report, the test material stability under storage conditions was unknown. Therefore, the concentration of AAD-1 protein of 63.5% in the powdered test material following storage has not been confirmed. The GMO Panel noted that haematology and clinical chemistry analyses were conducted on six mice/gender per group and coagulation (prothrombin time) was conducted on the remaining six animals in each group. The reasoning provided by the applicant was practical limitations in obtaining sufficient quantities of blood from mice for both haematology, clinical chemistry and coagulation examinations in the same animal. However, it is well known that when mice are used as the test animal, additional animals may be needed in each dose group, to conduct all required determinations. The GMO Panel also noted that the animals were not fasted prior to necropsy and blood collection, as recommended in the OECD TG 407. The AAD-1 protein group was statistically compared to the BSA control group; the latter was also compared to the vehicle control group in order to assess potential effects of the higher protein intake. All the parameters examined statistically were first tested for equality of variance using Bartlett s test (for sexes combined), and possibly scale-transformed if the test was significant. 40 Two different statistical analyses were then performed: a two-way analysis of variance (ANOVA; factors: sex and 38 Dossier: Part I Section D Dossier: Part I Section D7.8.1; additional information: 9/8/ a = EFSA Journal 2016;14(12):4633